The ultimate goal of this project is to develop a protocol for the cryopreservtion of Drosophila melanogaster embryos. We have investigated three different strategies: conventional cryopreservation, vitrification, and supercooling. Neither the conventional cryopreservation nor the supercooling procedure is appropriate for D. melanogaster embryos because of their subzero chilling sensitivity. By using a vitrification procedure, which precludes ice formation in the system and allows for cooling at ultra-rapid rates, we have obtained 18.4 +/- 8.8% survival (based on hatching of eggs) following recovery from liquid nitrogen. In preliminary studies, 3% of the larvae developed into adults. The adults were fertile and produced an F1 generation. Our immediate objective is to optimize the culture conditions for larvae development, pupariation and adult eclosion. This will include additions of ecdysteroids, dopa, and dopamine to the culture medium to maximize sclerotization during larval development. Subsequently, a systematic analysis of the effect of the various steps in the current vitrification procedure on the frequency of pupariation will be conducted, with optimization of the loading and dehydration steps. Additional modifications of the vitrification procedure that will be considered to increase the percentage of embryos that develop into adults include (1) the use of other cryoprotectants and mixtures of cryoprotectants in the loading step and (2) the development of improved vitrification solutions and procedures for their use that reduce their toxicity. Our working hypothesis is that toxicity of the vitrification solution is a function of the osmotic potential of the solution rather than its concentration per se. Therefore, studies to determine survival as a function of the osmotic potential of the suspending medium and the formulation of vitrification solutions with a high (less negative) osmotic potential are proposed. For this, there is a considerable range in the osmotic potential of different glass-forming solutions that may be used. The glass-forming tendency and stability of the amorphous state of these solutions will be characterized using differential scanning calorimetry (DSC). Included in these studies will be an analysis of the kinetics of ice crystallization as a function of the cooling and warming rates. To establish the extent of dehydration required to effect vitrification of the cytosol, the osmometric behavior of the embryos in the presence of these solutions will be characterized and predicted for various loading/concentrating/unloading protocols using computerized video image analysis in conjunction with irreversible-thermodynamic modelling. In addition, the phase behavior and kinetics of ice crystallization in the cytosol will be characterized by DSC. Systematic studies of the chilling sensitivity of the embryos will be conducted to determine the effects of temperature, time at a given temperature, cooling/warming rate, and the presence of cryoprotectants on the ability of embryos to survive subzero temperatures. When an appropriate level of survival (10 to 20% recovery of adults) is attained, we will determine viability after long-term storage (time scales of months), assess their genetic stability, and test the cryopreservation procedure with other strains of Drosophila. For this project, a multidisciplinary team has been assembled and includes two cryobiologists (Steponkus and Myers), a Drosophila geneticist (Maclntyre), an engineer for modeling studies (Pitt), a biophysicist with considerable experience in cryobiology (Bronshteyn), and technicians trained in entomology (Gardner) and Drosophila genetics (Varak).